PROJECT SUMMARY Amyotrophic lateral sclerosis (ALS) is a mostly sporadic and invariably fatal paralytic disorder. ALS pathogenic mechanisms are elusive and its etiology is poorly understood, even if environmental exposures, including toxic metals, are thought to play a key role. Progresses in both therapy and prevention for ALS have been held back by the lack of clear biomarkers indicative of patients? disease progression and patients? central nervous system (CNS) exposure to environmental toxicants. Here, we propose to test in two complementary U.S. cohorts whether the cargoes of blood extracellular vesicles (EVs), i.e., tiny membrane-bound compartments that shuttle biomolecules out of the CNS, can serve as novel biomarkers of CNS metal exposure and disease progression in ALS. EVs carry nucleic acids (including microRNAs [miRNAs]), proteins, and other elements are circulated throughout the body for inter-cellular and -organ communication. EVs also operate as ?trash bags? allowing cells to eliminate excesses of unwanted cellular materials, including toxic metals and proteins. CNS- EVs can be isolated from blood because they express on their surface the CNS-specific protein L1CAM. Blood CNS-EVs could open a direct window of observation into the ALS brain from the periphery and provide different types of disease- and exposure-relevant biomarkers. First, CNS-EVs may reflect disease progression by carrying pathogenic proteins progressively accumulating in ALS CNS tissues, such as the TAR DNA- binding protein 43 (TDP-43), or by showing progressive changes in miRNA profiles. Second, CNS-EV metal levels could be a direct surrogate of CNS metal load, and as suggested by metals? effects on miRNA profiles, they could provide a molecular fingerprint of patient metal exposure, when direct measurement is not possible. To test these hypotheses, we will leverage unique biospecimen collections from two well-phenotyped cohorts of ALS patients: (1) the COSMOS multi-center cohort (n=269 patients; 2-6 serial visits; blood); and (2) the US National Biorepository (UNB) pilot study (n=200; 2 visits; metal-free blood; CNS tissues for 19 patients). Each visit included clinical stage assessment through the ALS Functional Rating Scale-Revised. Our specific aims are to: Aim-1: Assess whether TDP-43 level in blood CNS-EVs is a biomarker of ALS progression by ELISA quantification of the TDP-43 content of L1CAM-EVs from the serial COSMOS and UNB blood draws, and testing whether TDP-43 changes are associated with ALS progression and survival. Aim-2: Investigate metal content in blood CNS-EV as a biomarker of patient?s exposure by measuring in the UNB CNS-EVs the levels of 6 metals linked to ALS, comparing them to paired CNS tissue levels, and testing their association with ALS progression. Aim-3: Test whether miRNA profiles in blood CNS-EVs are biomarkers of both ALS progression and metal exposure by sequencing CNS-EV miRNAs and testing if they mediate TDP-43?s and metals? effect on ALS progression. The novel and minimally invasive EV biomarkers tested here could greatly improve the evaluation of ALS treatment efficacy, and revolutionize metal exposure assessment.